Genetic alterations and allele frequency of BRAF V600E and TERT mutation in papillary thyroid carcinoma with intermediate-to-high recurrence risk: a retrospective study

The predictive value of allele frequency (AF) of BRAF V600E and TERT mutations in papillary thyroid carcinoma (PTC) remains controversial. We aimed to investigate the AF of BRAF V600E and TERT mutations in intermediate-to-high risk PTC and their association between tumor invasiveness, prognosis, and other mutations. Probe hybridization capture and high-throughput sequencing were used to quantitatively test 40 gene loci in 94 intermediate-to-high recurrence risk PTC patients, combined with clinical characteristics and follow-up for retrospective analysis. BRAF V600E mutation AF was linked to a increased risk of thyroid capsule penetration, recurrence, and concurrent mutations. Concurrent mutations could lead to a worse prognosis and increased invasiveness. TERT promoter mutation frequently accompanied other mutations and resulted in a poorer prognosis. However, there was no clear association between the TERT mutation AF and tumor invasiveness or recurrence. The sensitivity and specificity of predicting recurrence in intermediate-to-high risk PTC with BRAF V600E mutation AF > 28.2% were 60 and 80%. Although genetic alterations in PTC can differ among different ethnicities, the AF of BRAF V600E and TERT mutations may be similar. The AF of BRAF V600E has the potential to be a novel indicator in predicting PTC invasiveness and prognosis.


Introduction
In recent decades, there has been a global increase in the occurrence of thyroid cancer [1].Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer, and the BRAF V600E mutation is the most frequent genetic mutation in PTC [2].The BRAF gene encodes B-Raf protein kinase, which is a crucial member of the MAPK signaling pathway that regulates cell proliferation and differentiation [3].Research suggests an association between PTC with BRAF V600E mutation and tumor volume, invasiveness and metastasis [4,5].However, it remains controversial whether BRAF V600E accurately reflects the malignancy and prognosis of the tumor [6][7][8].The TERT gene encodes telomerase, which is a crucial enzyme for maintaining chromosomal telomere length.Activation of TERT gene mutations can sustain telomere length, leading to unlimited cell proliferation.Research has shown that mutations in the TERT gene are related to increased tumor size, invasiveness, lymph node metastasis and resistance to radiotherapy [9,10].However, the severity of PTC with BRAF V600E mutation can be variable, and patients with BRAF mutations may also present with different conditions, possibly related to the individual frequency of BRAF V600E in each patient [9].Allele frequency (AF) is calculated by dividing the number of mutated molecules by the total number of wild-type molecules at a specific position in the genome [11,12].In this retrospective review of gene test results from Chinese patients with intermediate-to-high recurrence risk PTC [13], we analyzed and explored the impact of BRAF V600E and TERT mutation AF, aiming to identify reliable indicators that reflect the invasiveness and prognosis of PTC.

Patients
A retrospective analysis was conducted on 106 patients with thyroid cancer who underwent genetic sequencing at the Department of Thyroid Surgery, The Second Hospital of Dalian Medical University, from November 2018 to December 2023.The inclusion criteria were: (1) postoperative pathology confirming PTC and (2) intermediate or highrisk stratification for PTC recurrence, stratified according to the ATA initial recurrence risk stratification [13].94 patients who met the inclusion criteria were included.The data collected included age, sex, stage, pathology, genetic testing results, and follow-up outcomes.

Second-generation sequencing
The gene testing was conducted by Beijing Genetron Health Co., Ltd.The Genetic testing loci was shown in Table 1.The steps of the testing process are as follows: (1) DNA Extraction: DNA concentration was measured using Qubit dsDNA HS Assay Kits (Thermo Fisher Scientific, Q32851), and RNA concentration was meas-ured using Qubit RNA HS Assay Kits (Thermo Fisher Scientific, Q32852).(2) Reverse Transcription and PCR Amplification: To prepare the reaction system, take 0.2-mL PCR tubes equal to the number of TNA samples + 2. Total TNA 100 ng, make up to 7 µL with nuclease-free water.For RNA heat treatment, use the following reaction conditions: 80 °C for 10 min followed by 25 °C for 3 min.Then add 1μL of reverse transcriptase solution and 2μL of reverse transcriptase buffer solution to the reaction, mix well, and place on ice.Finally, set the PCR machine program with synthesis conditions.Perform PCR amplification by taking out the cDNA amplification primer mix, specific adapter, and polymerase mix, melting, mixing and following the temperature and time conditions of 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min.(3) Library Construction: To mix the magnetic beads, place them at room temperature for 30 min, vortex to disperse the beads, and slowly aspirate the solution.Next, prepare an 80% ethanol solution.Add 3μL of DNA library from pool 1 to pool 2 in a 3:20 ratio, shake well, let it stand at room temperature for 5 min, centrifuge briefly, place on a magnetic stand for about 5 min until the liquid clears, and discard the supernatant.(4) Library Quantification: The library concentration should be quantified using the Qubit fluorescence quantification system with the recommended DNA nucleic acid quantification reagent kit.Additionally, the library concentration and band size should be quantified using Agilent 2100/2200/4200.A target fragment size of around 180-300 bp is recommended, and the dimer proportion should not exceed 25% to be considered qualified.Sequencing should be performed on samples using the GENETRON S5 gene sequencer produced by Genergy Bio Technology.The BaseCaller software is utilized for base identification and base information statistics, and the TMAP software is used to align the sequencing results to the human reference genome

Statistical analysis
The

Association between AF of BRAF V600E mutation and gender/age
The BRAF V600E mutation occurred in 75 cases (76.5%) of PTC patients, with a male-to-female ratio of  2, and the trend is illustrated in Fig. 1, indicating a rise-fall-rise pattern with peaks at ages 30-39 years and 70-79 years groups.According to ATA and AJCC guidelines [13], age 55 is a critical point for AJCC staging.Therefore, the study population was divided into ≥ 55 years and < 55 years groups, showing no statistically significant difference in BRAF mutation AF between the two groups (p = 0.28).

Association between AF of BRAF V600E mutation and extrathyroidal extension
Among PTC patients with BRAF V600E mutations, 18 cases (24%) showed extrathyroidal extension breaking the thyroid capsule, as shown in Table 3.The line graph in Fig. 2 shows the relationship between the number of cases with thyroid cancer invasion and the BRAF V600E mutation AF.
A significant association was found between extrathyroidal extension and the increased AF of BRAF V600E mutation.

Association between AF of BRAF V600E mutation and recurrence
Among PTC patients with BRAF mutations, 10 cases (13.3%) experienced recurrence, as shown in Table 3, with an average follow-up time of (1.86 ± 1.26) years.A significant association was found between BRAF V600E mutation AF and thyroid cancer recurrence (p = 0.023, OR = 10.080,OR (95%CI) = 1.010-1.154).Figure 2 shows the line graph depicting the recurrence rate/BRAF V600E mutation AF.The receiver operating characteristic (ROC) curve was created using sensitivity on the y-axis and (1-specificity) on the x-axis.The area under the curve (AUC) was 0.717 (95% CI = 0.541-0.893),as shown in Fig. 3.The point with the highest Youden index was at a BRAF V600E mutation AF of 28.2% (sensitivity = 60.0%,specificity = 80.0%, Youden index = 0.40, accu-racy=77.3%),as shown in Table 4. Table 5 shows that the risk of thyroid cancer recurrence is six times higher with a BRAF V600E mutation AF > 28.2% compared to AF ≤ 28.2%.

Discussion
The incidence of PTC is higher in females than in males, with a ratio of 2-3 to 1.However, in patients with intermediate-to-high recurrence risk PTC, females are only 1.36 times than males, suggesting that more males develop intermediate-to-high risk PTC.Some studies suggested that male is considered one of the risk factors for lymph metastasis in thyroid cancer [14].We observed no significant differences in the allele frequencies of BRAF V600E and TERT mutations between males and females, which indicates that the severity of thyroid cancer in males may be associated with other factors.The occurrence rates of TERT mutations in thyroid cancer do not differ significantly among Asians, Europeans, and Americans [9,10].However, the occurrence rate of BRAF V600E mutations varies significantly across different races.The incidence of BRAF V600E mutation varies among different populations.In American patients with PTC, the incidence is approximately 50.8% [3].In Korean patients, the incidence is around 72% [4], while in Japanese patients, it is about 38% [5].In Italian patients, the incidence is around 38.1% [7], and in Saudi Arabian patients, it is about 59.5% [6].In Chinese PTC patients, the incidence is approximately 72.4% [11], which is similar to the rate observed in our retrospective analysis (76.5%).Although BRAF V600E is considered a major driver in Chinese PTC populations, BRAF V600E AF in Chinese PTC patients is not significantly higher than in other regions and is even lower in our study.The reported BRAF V600E AF in PTC in other regions is approximately 26% in Canada [12] and around 27% in Italy [15].While our study found that the AF of BRAF V600E in Chinese patients was approximately 19.36%, demonstrating inconsistency between its AF and incidence characteristics.Gene mutations and fusions often do not occur simultaneously, exhibiting mutual exclusivity.Only 1 patient was found to have both TERT mutation and ETV6-NTRK3 fusion, but this patient's condition was not severe.Among the patients analyzed, the most common gene fusion was CCDC6-RET fusion (63.6%), followed by NCOA4-RET fusion (18.2%) and ETV6-NTRK3 fusion (18.2%), which may be related to a history of neck exposure [16,17].
The AF of BRAF V600E mutation is suspected to be associated with metastasis in PTC, leading to higher tumor staging and poor outcome [5,11,12].However, some studies have contradicted this claim, stating that the AF of BRAF V600E mutations does not significantly impact the prognosis and invasiveness of PTC [15,18].These discrepancies may be related to the diversity of populations in different countries or variations in the risk stratification of patients.Tumors form due to genomic instability in somatic cells, which can lead to the emergence of aggressive clones that can survive and outcompete other cells in the microenvironment.With the competition among cells, various genomic compositions (Allele Frequency) have emerged.The overall density of somatic mutations is relatively low, and this is considered the biological basis for the indolent clinical behavior observed PTC [18][19][20][21].The efficacy of AF varies in different literature, which may be due to its unclear efficacy in low-risk thyroid cancer patients, while its effect becomes more distinct in intermediate-to-high-risk patients, clearly demonstrating its association with the disease.Therefore, we focused on intermediate-to-high-risk population to avoid interference from the large low-risk PTC population.In addition, genetic testing is a costly procedure, and although we found that BRAF V600E AF can predict patient prognosis, it is apparently unnecessary for low-risk PTC patients, since next generation sequencing techniques are mainly applicable to populations with intermediate-to-high risk recurrence patients.However, as this is a single-center, single-region, single-ethnicity study, it has certain limitations.Future research could include multiple populations, regions, and larger sample sizes to further investigate the overall impact of BRAF and TERT gene frequencies on disease.In addition, 94.1% of TERT mutation carriers had other types of genetic alterations, which may be due to the small sample size of TERT mutations.The lack of significant association between TERT promoter mutation AF and disease prognosis may be influenced by other mutation types, which requires further investigation.
We analyzed the impact of BRAF V600E mutation AF on tumor invasion, co-occurring mutations, and recurrence in patients with PTC in intermediate and high-risk recurrence categories.For each 1% increase in BRAF V600E mutation frequency, the risk of tumor invasion, recurrence, and co-occurring gene mutations increases by 10.0%, 8.0%, and 8.8%, respectively.The combination of BRAF mutation with other gene mutations significantly increases the risk of thyroid cancer invading the thyroid membrane and recurrence, with TERT mutations being the most common co-occurring mutation.When the frequency of the BRAF V600E mutation is greater than 28.2%, the risk of thyroid cancer recurrence increases sixfold compared to when the frequency is 28.2% or lower.Using AF of BRAF V600E mutation > 28.2% to predict the recurrence of intermediate and high risk PTC has a sensitivity of 60% and a specificity of 80%, making it a potential new indicator for predicting the risk of thyroid cancer recurrence.
The study indicates that BRAF mutations are the primary genetic mutation events in PTC, followed by TERT mutations, while other types occur less frequently.TERT mutations are associated with older age (average 61.41 years) and a higher AF (average 46.71%).Research indicates that TERT mutation is infrequent in children and teenagers [22], while combined with BRAF mutation, they contribute to increased malignancy in thyroid cancer [23,24].Our study suggested that TERT mutations are often accompanied by other mutations.The higher malignancy of TERT mutations in PTC may be due to the co-occurrence of other mutations [25].The co-occurrence of BRAF mutations with TERT mutations is the most common scenario, and there is a positive correlation between the AF of BRAF V600E and the risk of co-occurring TERT mutations.According to extensive data reviewed by scholars, there appears to be no significant correlation between BRAF mutation and distant metastasis in PTC patients, while TERT mutation has been associated with distant metastasis.But our study identified a potential association between a high allele frequency of BRAF V600E and TERT mutation, which is consistent with the findings of previous research, BRAF mutations may lead to abnormal overexpression of the TERT promoter [26].However, among the 94 patients we reviewed, none showed evidence of distant metastasis.Further research is needed to explore the relationship between BRAF, TERT mutations and distant metastasis [27].

Conclusions
Although the incidence of BRAF V600E mutation varies across different regions, the AF of BRAF V600E mutation is similar between Asian and Western patients.AF of BRAF V600E is positively correlated with invasiveness and the risk of recurrence.It may induce other gene mutations, such as TERT mutations, thereby enhancing the invasive capabilities of the tumor and leading to a poorer prognosis.The AF of BRAF V600E shows potential as a novel indicator for predicting tumor invasiveness and prognosis.

between AF of TERT mutation and clinical statistics
TERT promoter mutations were present in 17 cases (17.3%) of PTC patients, with a gender ratio of 1:1.13 and an average age of (61.41 ± 10.86) years.The average TERT mutation frequency was (46.71 ± 11.09) %, and all mutations were

Table 4
Sensitivity and specificity of BRAF V600E mutation AF for predicting recurrence at various cutoff values (n = 75)

Table 8 .
There were no clear statistically significant differences in gender, age, thyroid capsule penetration, and recurrence among different AF of TERT mutation.
Gene fusion11 cases (11.22%) were detected with gene fusions, with a sex ratio of 1:1.75 and a mean age of (38.09 ± 16.15) years.The gene fusions included 7 cases of CCDC6-RET fusion, 2 cases of NCOA4-RET fusion, and 2 cases of ETV6-NTRK3

Table 6
BRAF monoallelic mutation group Versus BRAF combined with other mutations group (n = 75)

Table 9
Clinical characteristics of patients with gene fusion (n = 11)